title: infected_engorged_2_lib03359
description: Protocols: Colonies of O. moubata were maintained in the insectary within the biosafety level 2 facilities. All developmental stages were kept in darkness in an incubator (25°C; 85% RH). An artificial feeding system was applied to fed the ticks in vitro. To obtain non-infected unfed females, unfed adult O. moubata which had not fed for six months were used. To obtain non-infected engorged females, unfed adult O. moubata were allowed to fed to repletion as described above. Swine blood of the same lot number was used to obtain B. duttonii-infected females, except that the blood was spiked with in vitro cultured B. duttonii at a final concentration of 2 × 106 spirochaetes/ml, which corresponds to the spirochaetemia in the blood of an infected BALB/c laboratory mice during the first few days of infection. Tick midguts were dissected from unfed females and from fed females 24 h post feeding. Washed midguts were snap frozen in liquid nitrogen and cryofractured individually using the cryoPREP impactor (Covaris, Brighton, UK). Total RNA was isolated from the pulverized midgut by adding 750 µl TRIzol LS Reagent (Life Technologies, Darmstadt, Germany) and 250 µl chloroform. After centrifugation at 13,000 × g (4°C) the upper aqueous phase was collected and used as input for the Agencourt RNAdvance Tissue Kit (Beckman Coulter, Krefeld, Germany) in combination with a KingFisher Flex System (Thermo Fisher Scientific, Darmstadt, Germany) for automated extraction. A DNAse I (Qiagen, Hilden, Germany) digestion was included prior to final elution. After that, an appropriate amount of ERCC spike-in mix (Life Technologies) was combined with 2 µg total RNA for extraction of the poly(A) RNA fraction using the Dynabeads mRNA DIRECT Kit (Invitrogen, Carlsbad, USA). Fragmentation and construction of the strand specific whole transcriptome library was done using the Ion Total RNA‐Seq Kit v2.0 (Life Technologies, Carlsbad, USA).